Stabilized polyribonucleotide coding for an elastic fibrous protein

ABSTRACT

The invention relates to a polyribonucleotide, a cosmetic and pharmaceutical compound that has the polyribonucleotide and a medicinal product that has the polyribonucleotide and the compound.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of copending international patentapplication PCT/EP2014/056233 field on 27 Mar. 2014 and designating theU.S., which has been published in German, and claims priority from theGerman patent application DE 10 2013 005 361.7, filed on 28 Mar. 2013.The entire contents of these priority applications are incorporatedherein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains references to nucleic acid sequences and/oramino acid sequences which have been submitted concurrently herewith asthe sequence listing text file “5402P503_ST25.txt”, file size 60KiloBytes (KB), created on 23 Sep. 2015. The afore-mentioned sequencelisting is hereby incorporated by reference in its entirety pursuant to37 C.F.R. §1.52(e)(5).

FIELD

The present invention relates to a polyribonucleotide, a cosmetic andpharmaceutical composition comprising said polyribonucleotide, and amedical product comprising said polyribonucleotide or said composition.

BACKGROUND

Elastic fibers are the largest structures of the extracellular matrix.They give elastic properties to the tissue. The elastic fibers consistof two morphologically distinct components. The first and largestcomponent of the mature fiber is the elastin. The second component arethe micro-fibrills which mainly consist of fibrillin and are associatedwith further proteins such as the micro-fibrills associatedglycoproteins (MAGPs), fibulines, and theelastin-micro-fibrills-interface localized proteins (EMILIN). The lysyloxidase (LOX) is involved in the cross-linking of the elastic fibers.

Elastin and its soluble precursor tropoelastin belong to the majorstructural proteins of the body. It provides structure and support tothe connective tissue and is responsible for the elasticity of arteriesand the lung.

Elastin is encoded by the ELN gene. Mutations in the ELN gene may resultin inherited disorders such as dermatochalasis, Williams-Beurensyndrome, and sub valvular innate aortic stenosis (SVAS).

Arteriosclerotic blood vessels are subject to a loss of elastin whichcannot be naturally compensated by cells involved in the regeneration.This is due to the fact that elastin-forming cells only synthesize andsecrete new elastin up to a certain age and during the growth of theorganism. In the following the protein is cross-linked in theextracellular space with each other and with other proteins of theconnective tissue. Since elastin is particularly durable because of thecross-linking after having reached the full body height the need of anorganism is fulfilled and the synthesis is almost ceased.

Because of the reduced synthesis of elastin in the old age the skinloses its flexibility and begins to develop wrinkles.

There are no satisfying or even causal therapies of a deficientsynthesis of elastic fibrous protein, in particular elastin.

Hirano et al. (2007), Functional rescue of elastin insufficiency in miceby the human elastin gene: implications for mouse models of humandisease, Circulation Research 101: 523-531, describe the introduction ofhuman elastin by means of a DNA plasmid into mice oocytes. However, thisapproach is not suitable for a therapeutic application in humans.

SUMMARY

Against this background it is an object of the present invention toprovide a substance by means of which the problems mentioned at theoutset can be solved. In particular, such a substance should be providedwhich can counteract a lack of elastic fibers or elastic fibrousprotein, respectively, and which can stimulate the synthesis of elasticfibers or elastic fibrous protein.

This object is achieved by the provision of a polyribonucleotideencoding an elastic fibrous protein comprising a nucleotide sequencewhich comprises at least one chemical modification stabilizing saidpolyribonucleotide.

The inventors have surprisingly realized that a deficiency of thesynthesis of elastic fibrous protein can be counteracted in a causalmanner by providing the coding sequence to the cell in a form ready fora direct translation. The polyribonucleotide according to the inventioncan be introduced into the cells to be regenerated and can induce thesynthesis of elastic fibrous protein in situ. The cells transfer thesynthesized elastic fibrous protein into the natural path of theassembly of elastic fibers. This ensures not only the synthesis of theelastic fibrous protein but also the new synthesis of the elasticfibers. Neither the administration of elastic proteins as such nor ofother proteins being involved in the genesis of elastic fibers could sofar provide similar results.

The stabilization of polyribonucleotides, for example of mRNA, isextensively described in the state of the art. It is referred to thefollowing publications: US 2009/0093433, WO 2011/012316, WO 2012/135805,WO 2012/045082, WO 2012/019168, WO 2012/045075, WO 2012/158736.Furthermore, the stabilization of mRNA by chemical modification ofribonucleotides is described in the publications of Warren et al.(2010), High efficient reprogramming to pluripotency and directeddifferentiation of human cells with synthetic modified mRNA, Cell StemCell 7, p. 618 to 630, and Kormann et al. (2011), Expression oftherapeutic proteins after delivery of chemically modified mRNA in mice,Nature Biotechnology, Vol. 29, No. 2, p. 154 to 159, Mandal and Rossi(2013), Reprogramming human fibroblast to pluripotency using modifiedmRNA, Nature Protocols, Vol. 8, No. 3, p. 568 to 582.

Methods for the synthesis of polyribonucleotides, such as mRNA, areextensive described in the state of the art. One of the suitable methodsis the in vitro transcription (IVT). The resulting mRNA is also referredto as IVT-mRNA.

The inventors have realized that the synthesis of elastic fibrousprotein in situ, that means in the cell, can compensate a deficiency,e.g. due to a mutation but also due to an age-related cease of thenatural synthesis, in a targeted manner.

Because of the chemical modification the polyribonucleotide issufficiently stable to be translated by the cell-own machinery. However,the polyribonucleotide according to the invention is sufficientlyinstable to only develop a temporary effect so that side effects can belargely avoided.

As the inventors were able to show, the immune response of an organismtreated with the polynucleotide according to the invention issufficiently smaller than by using a reference polyribonucleotide whichis not chemically modified. This results in an additional increase inthe therapeutic benefit of the polyribonucleotide according to theinvention.

The object underlying the invention is herewith completely solved.

According to the invention it is preferred if the elastic fibrousprotein is selected from the group consisting of: elastin/tropoelastin,fibrillin, micro-fibrills associated glycoprotein (MAGP), fibuline,elastin-micro-fibrills-interface localized protein (EMILIN) and lysyloxidase (LOX) and precursors thereof.

This measure has the advantage that the polyribonucleotide according tothe invention is configured for the induction of the in situ synthesisof the most important elastic fibrous proteins. For the mentionedelastic fibrous proteins preference is given to the human variants sothat the use in humans is fostered.

In an embodiment the polyribonucleotide is an mRNA.

This measure has the advantage that the polyribonucleotide is providedin a form which can immediately be used by the cell-own proteinsynthesis machinery. This may include e.g. an in vitro transcribed mRNA(IVT-mRNA).

According to the invention one of the following mRNA stabilized bychemical modification is preferred: human elastin, transcript variant 1(cDNA, NCBI data base NM_000501.2; SEQ ID No. 2), transcript variant 2(cDNA, NCBI data base NM_001081752.1; SEQ ID No. 3), transcript variant3 (cDNA; NCBI data base NM_001081753.1; SEQ ID No. 4), transcriptvariant 4 (cDNA, NCBI data base NM_001081754.1; SEQ ID No. 5),transcript variant 5 (cDNA, NCBI data base NM_001081755.1; SEQ ID No.6).

In another embodiment the at least one chemical modification stabilizingsaid polyribonucleotide comprises a chemically modified nucleoside,preferably a modified uridine and/or cytidine.

By this measure the inventors have made use of the findings which aree.g. described by Kormann et al. (I.c.), namely that the mRNA after achemical modification of the uridine ribonucleotides and/or cytidineribonucleotides is not so easily recognized by structures of the immunesystem, such as the signal transduction mediating PRRs (“patternrecognition receptors”) or “Toll-like receptors”, thereby activating asignificantly weakened immune response and obtaining a longer half-lifeperiod.

According to the invention a nucleoside also encompasses a correspondingnucleotide comprising in comparison to the nucleoside additionalphosphate residues.

The following chemically modified uridines or uridine ribonucleotidesare of particular suitability: pseudouridine, 2-thiouridine,5-methyluridine, 5-methyluridine-5′-triphosphate (m5U),5-idouridine-5′-triphosphate (15U), 4-thiouridine-5′-triphosphate (S4U),5-bromouridine-5′-triphosphate (Br5U),2′-methyl-2′-deoxyuridine-5′-triphosphate (U2′m),2′-amino-2′-deoxyuridine-5′-triphosphate (U2′NH2),2′-azido-2′-deoxyuridine-5′-triphosphate (U2′N3),2′-fluoro-2′-deoxyuridine-5′-triphosphate (U2′F) and combinationsthereof.

Especially suitable chemically modified cytidines or cytidineribonucleotides are: 5-methylcytidine, 3-methylcytidine, 2-thiocytidine,2′-methyl-2′-deoxcytidin-5′-triphosphate (C2′m),2′-amino-2′-deoxycytidine-5′-triphosphate (C2′NH2),2′-fluoro-2′-deoxycytidine-5′-triphosphate (C2′F),5-iodcytidine-5′-triphosphate (I5U), 5-bromocytidine-5′-triphosphate(Br5U), 2′-azido-2′-deoxycytidine-5′-triphosphate (C2′N3) andcombinations thereof.

According to the invention at least approx. 5%, further preferably atleast approx. 7.5%, further preferably at least approx. 10%, and highlypreferably at least 25% of the nucleosides or uridines and/or cytidinesare modified.

Even though at least approx. 50% or approx. 100% of the nucleosides oruridines and/or cytidines can be modified the inventors have realizedthat a modification of up to approx. 25% of the nucleosides issufficient for a stabilization of the polyribonucleotide according tothe invention and reduction of the immune response. This has theadvantage that the costs for the preparation of the polyribonucleotideaccording to the invention are significantly lower than for a 100%modification.

In another embodiment the chemical modification is selected from thegroup consisting of: 5 cap structure, preferably a 5′ guanine cap, poly(A) tail, a cap structure analog [anti-reverse cap analog (ARCA;3′O-Me-m⁷G(5′)ppp(5′)ppp(5′)G)], a strengthening of thetranslation-initiation sequence at the start codon AUG, e.g. by thesequence (CCCCGC)aucGagAUG.

By this measure an additional stabilization of the polyribonucleotideaccording to the invention is achieved in a beneficial manner.

In another embodiment the polyribonucleotide according to the inventioncomprises the sequence of SEQ ID No. 1, where at least approx. 5%,further preferably at least approx. 7.5%, further preferably at leastapprox. 10%, and highly preferably at least approx. 25% of the uridines,and/or where at least approx. 5%, further preferably at least approx.7.5%, further preferably at least approx. 10%, and highly preferably atleast approx. 25% of the cytidines are chemically modified.

As mentioned above it is true that also at least approx. 50% or at leastapprox. 100% of the nucleosides can be modified, however an approx. 25%modification is sufficient.

The definition of “chemically modified” as set forth above applies herecorrespondingly. Preferably an exchange of uridine (U) againstpseudouridine or pseu-douridinetriphosphate (ΨFUTP) and/or of cytidine(C) against 5-methylcytidine or 5-methylcytidinetriphosphate (mCTP)takes place.

The polyribonucleotide according to the invention preferably encodes anamino acid sequence which is selected from the group consisting of: SEQID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11.

This measure has the advantage that the polynucleotide according to theinvention encodes one of the different isoforms of elastin (SEQ ID No.7: isoform a, NCBI data base NP_00049.2; SEQ ID No. 8: isoform b, NCBIdata base NP_001075221.1; SEQ ID No. 9: isoform c, NCBI data baseNP_001075222.1; SEQ ID No. 10: isoform d, NCBI data base NP_001075223.1;SEQ ID No. 11: isoform e, NCBI data base NP_001075224.1). The elastinisoforms comprise the effect which has been realized by the invention.

According to the invention it is preferred if the polyribonucleotide isconfigured for the induction of the synthesis of elastic fibrousproteins, in particular in age-related loss of elasticity of the skin(wrinkle formation), promotion of the wound healing, for the treatmentof a deficient synthesis of elastic fibrous proteins, or for thetreatment of a disease selected from the group consisting of:arteriosclerosis, aortic stenosis, aortic aneurysm, pulmonary emphysema,dermatochalasis, Williams-Beuren syndrome, sub valvular innate aorticstenosis (SVAS).

In addition, by means of the polyribonucleotide according to theinvention the vaginal tissue can be treated, e.g. following a pregnancy,for the stimulation of the elastin synthesis and the recovery of theelasticity.

Furthermore, the polyribonucleotide according to the invention can beconfigured for dental applications, for example for the reconstructionand/or regeneration of soft or hard tissues of the periodontium. Forthis reason the polyribonucleotide according to the invention ispreferably provided or configured for a transdermal application.

These measures have the advantage that the polyribonucleotide accordingto the invention is configured for the treatment of important diseasesand phenomena which may result from a reduced synthesis of elasticfibrous protein.

For this reason the invention relates to the use of thepolyribonucleotide according to the invention for the before-mentionedpurposes.

Furthermore, the invention relates a method for the induction of thesynthesis of elastic fibrous protein, in particular for thebefore-mentioned purposes, comprising the following steps: (1) provisionof the polyribonucleotide according to the invention, if applicable, ina pharmaceutically/cosmetically acceptable formulation, and (2)administration of the polyribonucleotides in or to an organism.

The application of the polyribonucleotide in or to an organism may beeffected via a topical application, for example onto the skin of theorganism, preferably the human skin. For this purpose the polynucleotidemay be a component of a dermatological dosage form such as a cream,lotion, ointment etc. However, the administration can also be effectedvia appropriate dosage forms systemically, orally, intravenously,intraarterially, intramuscularly, intrathecally, subcutaneously,intraperitoneally, intracardially, intravitreally, or intraosseouslyetc.

The transdermal administration of the polynucleotide according to theinvention can be effected by means of micro needles, nanopatches,nanoparticles or by means of a gene gun. In addition, active systems areappropriate which use iontophoresis. Here a very small electricalcurrent is transferred through the skin, which carries chargedmolecules. An example for this is the iontophoresis LTS-TTS system ofthe company LTS Lohmann Therapie-Systeme AG, Andernach, Germany.

Against this background a further object of the present invention is acomposition comprising the polyribonucleotide according to theinvention. The composition according to the invention may be acosmetical and/or pharmaceutical composition comprising a cosmeticallyor pharmaceutically acceptable formulation. Cosmetically andpharmaceutically acceptable formulations are generally known in thestate of the art. They are e.g. described in the assay of Kibbe et al.,Handbook of Pharmaceutical Excipients, 5. Edition (2006), AmericanPharmaceutical Association. The compositions may be configured as a monopreparation which contains the polyribonucleotide as the only activeagent. However, they may contain additives and, if applicable, furtheractive agents and excipients which are beneficial for the uses accordingto the invention, including transfection tools such as liposomes, hydrogels, kationic polymers or peptides, salts, binding agents, solvents,dispersing agents and further compounds which are commonly used inconnection with the formulation of cosmetics and pharmaceuticals.

In another embodiment of the invention the composition can additionallycomprise an immuno suppressive agent, preferably an interferoninhibitor.

This measure has the advantage that due to the chemical modification thealready reduced immune response of a host treated with thepolyribonucleotide according to the invention is further reduced. Thesuppression of the immune response after the administration of atherapeutic mRNA by the use of the interferon inhibitor B18R which issuited for the use according to the invention, is documented in thestate of the art, for example in Warren et al. (I.c.).

Another subject-matter of the present invention is a medical product,for example a patch or implant, which comprises the polyribonucleotideaccording to the invention or the composition according to theinvention, respectively, or which is coated with the latter. The implantmay be a medical implant, preferably a stent including a coronary stent,or vascular implant, stent graft or a bone implant.

The implant allows a targeted treatment of arteriosclerotic bloodvessels and/or local tissue areas, such as vaginal tissues, soft andhard tissues of the periodontium for the recovery of the elasticity.

Another subject-matter of the present invention is a wound dressingcoated with the polyribonucleotide according to the invention.

Such a wound dressing can reduce the scaring and maintain the elasticityof the scar tissue by the induction of elastin synthesis.

It is to be understood that the features of the invention mentionedabove and those yet to be explained below can be used not only in therespective combination indicated, but also in other combinations or inisolation, without leaving the scope of the present invention.

The present invention is now further explained by means of embodimentswhich result in further characteristics and advantages of the invention.The examples are purely illustrative and do not restrict the scope ofthe invention. Reference is made to the enclosed figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Plasmid preparations of four individually selected bacteriacolonies were tested for correct insert by means of ELN-specific PCR.Detection of PCR products by means of agarose gel electrophoresis, 1.2%(120 V, 30 min). PCR cycles: denaturating 3 min, 94° C., 1. 40 sec, 94°C.-2. 1 min, 58° C.-3. 2.5 min, 72° C.; final amplification: 7 min, 72°C.;

FIG. 2: Distinctively cutting restriction enzymes were determined bymeans of test digests of the plasmid and analysis in the following on a1% agarose gel (120 V, 40 min). Bands: 1-marker/DNA ladder, 2-5: testdigest of the plasmid with different downstream (3′) cutting restrictionenzymes: 2: Not I, 3: Xba I, 4: Bfa I, 5: Xho I, 6: digest withdownstream (5′) cutting enzyme, EcoRI, 7-10: downstream cutting enzymeswere combined with Ecor I (double digest), order as 2-5. kb=kilobases;

FIG. 3: In vitro synthesized ELN-mRNA in denaturating agarose gelelectrophoresis 1% (16% formaldehyde, 100 V, 45 min). 1: IVT reactionafter 3 h incubation; 2: IVT reaction with polyadenylating mix and 25min incubation; 3: as in 2, after 45 min; 4: marker; 5-6: purified,polyadenylated ELN-mRNAs; 5, 6: with Ambion MEGAclear Kit (catalognumber AM1908); 7: with Qiagen RNeasy Midi Kit, double RNA amount(catalog number: 75142);

FIG. 4: Detection of reporter gene expression: detection of theLuciferase expression after transfection of IVT-Luciferase-mRNA intoEA.hy926. hpt=hours post transfection; dpt=days post transfection;

FIG. 5: In vitro synthesized ELN protein on the basis of four differentin vitro synthesized ELN-mRNAs and controls: 1, 2: marker; 3-6: proteinsynthesis with ELN-mRNAs from different in vitro transcription reactions(5: mRNA with modified nucleotides); 7: in vitro translation reactionwithout mRNA (negative control); 8: as in 7, but with addition of human,soluble elastin protein from Calbiochem (catalog number: 324751) afterincubation time; 9: only water solved elastin protein fromCalbiochem—according to data sheet the protein should run as a smearband between 5 and 60 kDa. Native elastin protein is detected by meansof the used antibody at a level of 50 kDa.

FIG. 6: Detection of the elastin expression by measuring the amount oftropoelastin in EA.hy926 cells after transfection at three successivedays with IVT-ELN-mRNA; they contained different fractions of modifiednucleic acids (mCTP/ΨFUTP) and Lipofectamin2000. RNA or Lipofectamin2000alone were used as negative controls.

FIG. 7: Detection of the elastin expression in the supernatant of theEA.hy926 cells after transfection with 100% modified (mCTP/ΨFUTP)IVT-ELN-mRNA by means of dot-blot assays; M: only cell culture medium,M+L: cell culture medium plus transfection reagent.

FIG. 8: Immune activation of EA.hy926 cells after the transfection ofIVT-ELN-mRNA; the expressions were normalized to the housekeeping geneGAPDH. The negative controls were set to 1.

FIG. 9: Detection of the reporter gene expression: microscopic images of“stented” vessels, focus on the border between stent and tissue. A, B:negative controls (uncoated stents). C-E: eGPF-mRNA-coated stents. Thesize of the images corresponds to an area of 30×30 μm. Upper picture:original image. Lower picture: images with FIDSAM and contrastcorrection.

FIG. 10: Detection of the Luciferase expression in pig skin aftertransfection with Luciferase mRNA

EXAMPLES 1. Plasmids Constructs for the RNA Synthesis

An elastin, transcript variant 1 encoding (SEQ ID No. 2) Sp6-promotercontaining plasmid, pCMV-Sp6_ELN, cloned into E. coli, was purchasedfrom Thermo Scientific.

The Luciferase encoding, T7-promoter containing plasmid, pCMV-GLuc-1used as a reporter gene, was purchased from Nanolight Technology, Inc.and cloned into Qiagen EZ competent E. coli from the Qiagen PCT cloningkit.

The plasmids contain sites for sequencing with primers such as the M13forward and reverse primers, and promoter regions for polymerases, suchas Sp6 and T7. These sequences can be found 5 or 3′ to the insertedsequence of interest, respectively. Additionally, the insert region isflanked by short recognition sequences for specific restriction enzymes.

2. Verification of Plasmid Inserts

The plasmids were isolated with the Qiagen Plasmid Maxi kit (Qiagen).

Insert-specific primers were designed with a free primer designing toolfrom NCBI and produced by Eurofins MWG Operon. The plasmid inserts wereverified by PCR with the insert-specific primer pairs (FIG. 1), and thefollowing cycling parameters were used: denature 3 min at 94° C.; 1. 40sec at 94° C.; 2. 1 min at 58° C.; 3. 2.5 min at 72° C.; repeat steps1-3, 28×; final amplification 7 min, 72° C.

Additionally, plasmid inserts were sequenced completely through thecompany GATC Biotech with self-designed insert-specific primers and withM13-forward and reverse primers. The sequence assembly was done with theDNA-baser program.

3. In Vitro Synthesis of ELN-mRNA with Different Amounts of ModifiedNucleic Acids

For the in vitro transcription (IVT) plasmids were linearized downstreamof the gene of interest with the Fast Digest Enzyme System(Fermentas/Thermo Scientific) and purified with the MiniElute PCR cleanup kit (Qiagen). Test digests were performed prior to the experiment(FIG. 2).

The in vitro transcription was performed according to the manufacturer'sinstructions with MEGAscript Sp6 kit. For each 40 μL IVT reaction 1 μgof linearized template was used. To optimize the stability andcytocompatibility of IVT-mRNAs in the reactions different ratios of thenucleic acid triphosphates 5-methylcytidine and pseudouridine (TriLinkBiotechnologies) were combined with standard nucleotides from the kit.

TABLE 1 nucleotide mixtures in the IVT reactions with different amountsof 5-methylcytidine and pseudouridine 5-methyl- CTP citidine UTPPseudouridine 25% modification 5.25 mM 1.75 mM 5.25 mM 1.75 mM 50%modification  3.5 mM  3.5 mM  3.5 mM  3.5 mM 100% modification  —   7 mM—   7 mM

In each IVT reaction 6 mM ATP, 3 mM GTP (from the kit) and 2 mM3′-O-Me-m⁷G(5′)ppp(5′)G (anti-reverse cap-analog from New EnglandBiolabs) were used. Further components were added as indicated in themanual of the kit.

The reactions were incubated for 3.5 hours at 37° C. and treated withDNase I from the kit at the end of the incubation period in compliancewith the instructions of the manufacturer, in order to eliminate thetemplate DNA.

The polyadenylation was made with the PolyA Tailing kit (Ambion)according to the instructions of the manufacturer.

The mRNA was purified with the RNeasy mini kit (Qiagen). The detectionof the elastin mRNA was made by denaturating agarose gel electrophoresis(FIG. 3). The concentrations of mRNAs were measured by means ofBioPhotometer6131 (Eppendorf).

4. In Vitro Translation of In Vitro Transcribed mRNAs

The quality of the in vitro transcribed mRNA was determined indirectlyby in vitro translation with the Retic Lysate kit (Ambion), followingluminescence measurement for the Luciferase or western blot analysis forelastin. The in vitro translation reactions were set up according to theinstructions of the manufacturer.

5. Cell Lines and Chemicals Used for the Transfection of IVT-mRNAs

The lung carcinoma cell lines A549 and SK-MES were used for the firsttrials. The endothelial cell/A549-hybridoma cell line EA-hy926 was usedto establish effective transfection methods for endothelial cells andtissues.

All mRNA transfections were performed by using the transfection reagentLipofectamine2000 (Invitrogen/Life Technologies). The medium fortransfections was OptiMEM (Gibco/Life Technologies). The negativecontrols throughout all experiments were OptiMEM with equal amounts ofLipofectamine2000 used for mRNA transfections or mRNA withouttransfection reagent in OptiMEM.

6. Transfection of IVT-mRNAs in Cell Culture

For the transfections with elastin mRNA the cells were plated with adensity of 500,000 cells per well in 6-well plates one day prior to theexperiments.

The transfection was made with 5 or 10 μg of elastin mRNA and 3.3 μL oftransfection reagent per well diluted in OptiMEM, based on theindications of the manufacturer.

The medium with the mRNA transfection complexes was added to the cellsand the plates were incubated for 4 hours under cell culture conditions.Afterwards, ⅔ of the transfection mixes were replaced with fresh culturemedium and the cells were incubated overnight. This transfection methodwas repeated for the following 2 days. At the third day the transfectioncomplexes were completely replaced with fresh cell culture medium.

Medium and cells were analyzed by one day after the last transfection.

7. Expression of the Luciferase Reporter Gene In Vitro

The first assessment of the Luciferase expression was performed 5 hoursafter the transfection and then following every day until the expressiondeclined. For luminescence measurements, representing the Luciferaseexpression, 20 μL medium was taken from each well 6/24/48/72 hours and5/10/25 days after transfection, with medium change after each sampletaking.

8. Measurement of the Luciferase Activity

The activity of the Luciferase enzyme directly after the in vitrotranslation or 5 hours to 30 days after the transfection of IVT-mRNAinto the cells was assessed by adding 100 μL of 2.5 ng/μL substratecoelenterazin to 20 μL cell medium from transfected cells or in vitrotranslation reaction, respectively. The resulting luminescence of theprobes was measured in a microplate reader (Mithras LB 940, BertholdTechnologies). FIG. 4 shows the measured luminescence relating to theLuciferase expression in the medium of IVT-mRNA transfected EA.hy926cells.

The results show that even a low amount of 200 ng of IVT Luciferase mRNAcan induce a significant Luciferase expression even after a shortincubation period of 5 hours. A high expression can be reached with 1 μgof IVT-mRNA up to 24 hours after the transfection, however the followingexpression course does not differ from the probes with lower amount ofIVT-mRNA. Even another increase of the amount of transfected IVT-mRNA upto 2 μg does not result in a higher expression.

9. Detection of (Tropo-)Elastin by Western Blot

The proteins from the in vitro translation reactions were separated on a8% SDS-PAGE and blotted on a nitrocellulose membrane for theimmunodetection. The primary antibody was a rabbit polyclonal ELNantibody (central region) from Abgent and the secondary antibody was agoat anti-rabbit IgG (whole molecule) Alkaline Phosphatase Conjugatefrom Sigma-Aldrich. The elastin protein was revealed by precipitation ofthe indigo dye resulting from NBT/BCIP reaction with alkalinephosphatase (FIG. 5).

The detection of elastin after the in vitro translation made on thebasis of IVT-elastin-mRNA confirms the integrity of the mRNA accordingto the invention. It is understood that a protein detection can onlyoccur when the synthesized protein corresponds to the structures againstwhich the antibody has been developed. For this reason the mRNAaccording to the invention must have been present in its entirety andfunctionality for the protein synthesis. Although there is apparently anon specific detection of proteins existing in the in vitro translationmix the specificity of the elastin band is unambiguous since it does notexist in the negative control which only contains the in vitrotranslation mix without IVT-mRNA.

10. Detection of the Expression of Tropoelastin in the Cell Culture

The expression of elastin was analyzed with the Fastin™ Elastin Assay(Biocolor life science assays) according to the manufacturer'sinstructions. FIG. 6 shows the amount of tropoelastin isolated 24 hoursafter the last transfection with elastin-IVT-mRNAs, which containedvarious parts of modified nucleic acids.

After a 3-fold transfection of IVT-elastin-mRNA a significant expressionof tropoelastin, i.e. of soluble and non cross-linked elastin, could bedetected in the cells. It is clear that a particular large amount of 10μg of transfected IVT-ELN-mRNA has no increasing effect on the elastinexpression over only 5 μg. Also the higher amount of modifiednucleotides has no positive influence on the expression. Therefore itseems that 5 μg of the IVT-ELN-mRNA with 25% of modified CTP/UTP cancause a sufficient detectable expression of elastin.

In another experiment 3×10⁵ cells per well of a 6 well plate wereseeded. At the following day the supernatants were aspirated and thecells were washed with 1 ml PBS. Then an incubation took place for 4hours with Opti-MEM (M), Opti-MEM with Lipofectamine 2000 (M+L), andOpti-MEM with Lipofectamine 2000 and 2.5 μg of elastin-mRNA (100%5mCTP/ΨFUTP). The cell supernatants were collected after 24 and 48hours. The supernatants were analyzed by means of dot-blot. The resultis shown in FIG. 7. The detection of elastin was made by means ofelastin-specific AB. The cells with elastin-mRNA-incubation (M+L+elastinmRNA) show a significantly stronger staining than the cells withoutmRNA-transfection (M, M+L). The EA.hy926-cells without elastin-mRNAsynthesize low amounts of elastin, however by theelastin-mRNA-transfection the elastin synthesis is significantlyincreased.

11. Assessing the Immunogenicity of the IVT-mRNAs

Transfections for the analysis of cytokines and other markers of theimmune activation were performed according to the mRNA-transfectionsdescribed above. Additionally, the immunostimulantpolyinosinic/polycytidylic acid (Sigma-Aldrich) was transfected at 100ng/well as a positive control for cytokine activation. The cells wereincubated with the transfection mix under cell culture conditions andthe medium was replaced after 3 hours.

The following day, cells were lysed and the RNA was extracted with AurumTotal RNA Mini Kit (Biorad). The RNA-concentration was measured and 40ng of each sample was used for cDNA-synthesis with iScriptcDNA-synthesis kit (Biorad). The generated cDNA was used diluted in(qRT)-PCR reactions with the iQ SYBR Green Supermix (Biorad) intriplicates for each sample, combined with a specific primer pair forIFN-∀, IFN-γ, IL-1 B, IL-12, IL-6, IL-8, TNF-α and a GAPDH-specificprimer pair. For the quantification of the immune marker expressionlevels a qRT-PCR was performed in 96-well plates in the CFX ConnectReal-Time PCR detection system (Biorad) (FIG. 8). The results show thatthe in vitro synthesized elastin-mRNA does only cause a very lowactivation of cytokines in this highly sensitive assay.

12. Coating of Coronary Stents with eGFP-m RNA

In another experiment the in vivo expression of eGFP via IVT-mRNA,coated on coronary stents was examined. The in vitro synthesis of theeGFP-mRNA was effected with the plasmid construct pcDNA3.3_eGFP asdescribed in Warren et al. (I.c.). The plasmid was provided by theauthors via Addgene (Cambridge, Mass., USA).

BMS coronary stents 3×20 mm of Qualimed (Winsen, Germany) weredip-coated, in an emulsion of 70 μg in vitro transcribed eGFP-mRNAcomplexed with 20 μL of Lipofectamin2000 in nuclease free water and 150μg of polyactic-co-glycolic acid RESOMER® RG 502 H (Sigma Aldrich)solved in ethyl acetate.

The study was performed in accordance with the German animal welfare lawand the recommendations on the care and use of laboratory animalspostulated by the FELASA (Federation of European Laboratory AnimalScience Associations). All protocols and procedures were approved by theAnimal Care and Welfare Commission of the University of Tubingen.

For this study two female pigs of approx. 65 kg (German land race)supplied by a local “specific pathogen-free” (SPF) breeding facilitywere used for this study and included in the analysis. After arrival atthe animal facility of the University of Tubingen, all animals wereallowed one week of adaptation prior to the intervention. During thisperiod clinical examinations were carried out to ensure the healthstatus, especially in consideration of the cardiovascular system.

The stents were implanted via a balloon catheter (3 mm) into the leftand right coronary arteries of each pig and expanded. The location ofthe stents was displayed with help of an X-ray apparatus (C-Bogen) andradiopaque material. An overstretching of the arteries was provoquedintentionally. After the implantation, the animals received heparin andclopidogrel to prevent postoperative thromboses.

44 hours after the implantation of the coated stents, the pigs wereeuthanized. The “stented” vessels were isolated and fixed overnight in4% formaldehyde.

For the fluorescence analysis, the stents were embedded inmethylmetacrylate based embedding system Technovit® 9100 fromHeraeusKulzer (Wehrheim, Germany), and analyzed by fluorescenceintensity decay shape analysis microscopy FIDSAM (fluorescence intensitydecay shape analysis microscopy) at the Institute of AnalyticalChemistry of the University of Tubingen.

The result is shown in FIG. 9. There the part of fluorescent tissuevisible after subtraction of the autofluorescence is shown. Thisfluorescence is only due to the induced eGFP expression and thereforethe evidence for efficient uptake and translation of the IVT-mRNAencoding eGFP by cells surrounding the stent material.

13. Pig Skin Model

A pig skin model was established to detect the synthesis of themRNA-induced elastin in the skin. In the first experiments 2.5 μg ofLuciferase m RNA/Lipofectamin 2000 complexes were injected into theskin. The skin was chopped after 24 h and for isolating the Luciferasethe cells were lysed. The result is shown in FIG. 10. By means of theLuciferase Assay the successful transfection of the cells withLuciferase mRNA was demonstrated.

Sequences

SEQ ID No. 1: Nucleotide sequence of the IVT-elastin-mRNA (as used inthe embodiments)

SEQ ID No. 2: Nucleotide sequence of the cDNA, derived from the mRNA ofthe human elastin, transcript variant 1 (NM_000501.2)

SEQ ID No. 3: Nucleotide sequence of the cDNA, derived from the mRNA ofthe human elastin, transcript variant 2 (NM_001081752.1)

SEQ ID No. 4: Nucleotide sequence of the cDNA, derived from the mRNA ofthe human elastin, transcript variant 3 (NM_001081753.1)

SEQ ID No. 5: Nucleotide sequence of the cDNA, derived from the mRNA ofthe human elastin, transcript variant 4 (NM_001081754.1)

SEQ ID No. 6: Nucleotide sequence of the cDNA, derived from the mRNA ofthe human elastin, transcript variant 5 (NM_001081755.1)

SEQ ID No. 7: Amino acid sequence of the elastin isoform a [homosapiens] (NP_00049.2)

SEQ ID No. 8: Amino acid sequence of the elastin isoform b [homosapiens] (NP_001075221.1)

SEQ ID No. 9: Amino acid sequence of the elastin isoform c [homosapiens] (NP_001075222.1)

SEQ ID No. 10: Amino acid sequence of the elastin isoform d [homosapiens] (NP_001075223.1)

SEQ ID No. 11: Amino acid sequence of the elastin isoform e [homosapiens] (NP_001075224.1)

What is claimed is:
 1. Polyribonucleotide encoding an elastic fibrousprotein having a nucleotide sequence comprising at least one saidpolyribonucleotide stabilizing chemical modification. 2.Polyribonucleotide of claim 1, wherein the elastic fibrous protein isselected from the group consisting of: elastin/tropoelastin, fibrillin,micro-fibrills associated glycoprotein (MAGP), fibuline,elastin-micro-fibrills-interface localized protein (EMILIN), lysyloxidase (LOX), and precursors thereof.
 3. Polyribonucleotide of claim 1which is an mRNA.
 4. Polyribonucleotide of claim 1, wherein the at leastone polyribonucleotide stabilizing chemical modification comprises achemically modified uridine or cytidine.
 5. Polyribonucleotide of claim4, wherein the chemically modified uridine is selected from the groupconsisting of: pseudouridine, 2-thiouridine, 5-methyluridine,5-methyluridine-5′-triphosphate (m5U), 5-idouridine-5′-triphosphate(15U), 4-thiouridine-5′-triphosphate (S4U),5-bromouridine-5′-triphosphate (Br5U),2′-methyl-2′-deoxyuridine-5′-triphosphate (U2′m),2′-amino-2′-deoxyuridine-5′-triphosphate (U2′NH2),2′-azido-2′-deoxyuridine-5′-triphosphate (U2′N3),2′-fluoro-2′-deoxyuridine-5′-triphosphate (U2′F) and combinationsthereof.
 6. Polyribonucleotide of claim 4, wherein the chemicallymodified cytidine is selected from the group consisting of:5-methylcytidine, 3-methylcytidine, 2-thiocytidine,2′-methyl-2′-deoxcytidin-5′-triphosphate (C2′m),2′-amino-2′-deoxycytidine-5′-triphosphate (C2′NH2),2′-fluoro-2′-deoxycytidine-5′-triphosphate (C2′F),5-iodcytidine-5′-triphosphate (I5U), 5-bromocytidine-5′-triphosphate(Br5U), 2′-azido-2′-deoxycytidine-5′-triphosphate (C2′N3) andcombinations thereof.
 7. Polyribonucleotide of claim 1, wherein thechemical modification is selected from the group consisting of: 5 capstructure, preferably a 5′ guanine cap, poly (A) tail, a cap structureanalog [anti-reverse cap analog (ARCA; 3′O-Me-m⁷G(5′)ppp(5′)ppp(5′)G)],a strengthening of the translation-initiation sequence at the startcodon AUG by the sequence (CCCCGC)aucGagAUG.
 8. Polyribonucleotide ofclaim 1, comprising a ribonucleotide sequence where at least approx. 25%of the uridines, or where at least approx. 25% of the cytidines arechemically modified.
 9. Polyribonucleotide of claim 1, comprising thesequence of SEQ ID No. 1, where at least approx. 25% of the uridines, orwhere at least approx. 25% of the cytidines are chemically modified. 10.Polyribonucleotide of claim 1, encoding an amino acid sequence selectedfrom the group consisting of: SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9,SEQ ID No. 10, and SEQ ID No.
 11. 11. Polyribonucleotide of claim 1,configured for any of the following indications: the induction of thesynthesis of an elastic fibrous protein, in age-related loss ofelasticity of the skin (wrinkle formation), for promoting the woundhealing, for the recovery of the elasticity of vaginal tissue, soft andhard tissue of the periodontium.
 12. Polyribonucleotide of claim 1configured for the treatment of a deficient synthesis of elastic fibrousprotein.
 13. Polyribonucleotide of claim 1 configured for the treatmentof a disease selected from the group consisting of: arteriosclerosis,aortic stenosis, aortic aneurysm, pulmonary emphysema, dermatochalasis,Williams-Beuren syndrome, sub valvular innate aortic stenosis (SVAS).14. Cosmetic composition, comprising the polyribonucleotide of claim 1and a cosmetically acceptable formulation.
 15. Pharmaceuticalcomposition, comprising the polyribonucleotide of claim 1 and apharmaceutically acceptable formulation.
 16. Medical product comprisingthe polyribonucleotide of claim 1 or the composition of claim
 15. 17.Medical product of claim 16 which is coated with the polyribonucleotideof claim 1 or the composition of claim
 15. 18. Medical product of claim16 which is selected from the group consisting of: medical patch,vascular implant, and stent.